Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.
Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km 45,000 M-1s-1) comparable to purified human GranB, RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.
Xia
Z
Z
Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kam
C M
CM
Huang
C
C
Powers
J C
JC
Mandle
R J
RJ
Stevens
R L
RL
Lieberman
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003546:Cysteine Endopeptidases / Q000378:metabolism; D053938:DNA Fragmentation / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D053804:Granzymes; D007694:Killer Cells, Natural / Q000201:enzymology; D007700:Kinetics; D010455:Peptides / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology
D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases